Characterization of acidic Ca(2+)-independent phospholipase A2 of bovine lung.

An acidic Ca(2+)-independent phospholipase A2 (aiPLA2) has been isolated previously from rat lung and a human cDNA has been described. This study applied the method to larger scale isolation of the native protein from the bovine lung. A polyclonal antibody was generated to a 15 amino acid synthetic peptide based on a conserved rat/human sequence. This antibody recognized a single protein band with an estimated molecular mass of approximately 29 kDa in a soluble fraction obtained from bovine lung homogenate. A 29 kDa protein that reacted with the aiPLA2 antipeptide antibody was detected in fractions containing aiPLA2 activity on sequential column chromatographies. The partially purified enzyme showed 176-fold increase over the homogenate in Ca(2+)-independent PLA2 activity at pH 4. Activity was maximal with phosphatidylcholine substrate and was significantly less with phosphatidylethanolamine and anionic phospholipids. The enzyme had no acyl group preference in phosphatidylcholine and showed no preference for oxidized substrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiPLA2 activity was inhibited by a transition state phospholipid analog (MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine protease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacyl bromide, and ATP. Analysis of N-terminal amino acid sequence for the 29 kDa protein demonstrated its high homology to human 26 kDa aiPLA2. These was no significant change in molecular mass of the protein following treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA2 immunoreactivity with lamellar body, lysosomal, and cytosolic fractions. These results indicate isolation from bovine lung of a 29 kDa acidic Ca(2+)-independent phospholipase A2 homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholine.